This application seeks continued funding to investigate a remarkable aromatization reaction catalyzed by nikD, an unusual flavoprotein oxidase that plays a critical role in the biosynthesis of nikkomycin antibiotics. Nikkomycins are peptidyl nucleosides that act as antifungal agents by blocking the biosynthesis of chitin, the second most abundant polysaccharide in Nature. The nonribosomal peptide in nikkomycins contains an essential N-terminal pyridyl residue that is synthesized by nikD in a multistep reaction comprising two redox cycles, a dihydropicolinate intermediate (DHP), and at least one isomerization step. The nikD substrate, piperideine-2-carboxylate (P2C), is a labile compound that is derived from L-lysine in a reaction catalyzed by an L-lysine-"-aminotransferase (LAT). The three-dimensional structure of nikD, determined in the past period of support, reveals two distinct substrate binding modes, an "aromatic cage" that surrounds the ring of nikD ligands, and a mobile cation binding loop. We propose to evaluate the catalytic role of the "aromatic cage" and monovalent cations. We will determine whether P2C degradation under physiological conditions is prevented by direct transfer of the metabolite (substrate channeling) from LAT to nikD. Our recent biochemical studies indicate that nikD oxidizes P2C to a DHP isomer (DHPx) that cannot be directly oxidized to picolinate. Instead, a reduced form of nikD catalyzes the isomerization of DHPx to produce an isomer (DHPy) that can be converted to picolinate. There are six possible DHP isomers. We will establish the identity of the DHP intermediates. We will also evaluate a novel hypothesis that the reduced flavin cofactor in nikD acts as the acid-base catalyst that is required for DHP isomerization. Our multifaceted experimental strategy includes crystallographic studies, techniques to monitor protein-protein interaction, use of isotopically labeled P2C and alternate or potential suicide substrates, mutagenesis, incorporation of modified flavins, independent synthesis of a postulated DHP intermediate, small molecule structural studies, and use of a stopped-flow spectrophotometer to monitor ligand binding, electron transfer steps, and DHP isomerization. The mechanism underlying the nik D reaction will provide a paradigm for related enzymatic aromatization reactions that produce heteroaromatic functional groups that act as important pharmacophores in many other nonribosomal peptide antibiotics (e.g., streptogramin, chlorobiocin, pyoluteorin).